GENETIC CONTROL OF FETAL Y-GLOBIN IN ADULT RED CELLS

成年红细胞中胎儿 Y 珠蛋白的遗传控制

• 批准号: 3233757
• 负责人: JOHN G GILMAN
• 金额: $ 10.53万
• 依托单位: MONTEFIORE MEDICAL CENTER (BRONX, NY)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-20 至 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233757
• 关键词: DNA binding protein; DNA footprinting; binding proteins; chemical binding; chemical structure function; electroporation; erythrocytes; gene deletion mutation; gene expression; genetic enhancer element; genetic manipulation; genetic markers; genetic promoter element; genetic transcription; human population genetics; human tissue; molecular pathology; nucleic acid probes; nucleic acid sequence; plasmids; radiotracer; sickle cell anemia; thalassemia; tissue /cell culture; transfection

Elevated fetal hemoglobin (Hb F) ameliorates diseases of adult hemoglobin, such as sickle cell anemia (SS) and beta-thalassemia. This laboratory has demonstrated mutations associated with increased expression of gamma genes in vivo, at -158 (C to T ) and -161 (G to A) of the G gamma gene and -202 ( C to T) of A gamma. A 4 base pair deletion at -222 to -225 of the A gamma gene is associated with decreased A gamma expression in Mediterranean haplotype II betao-thalassemia. The Benin betaS haplotype is generally associated with low Hb F, but preliminary data show high Hb F determinants linked to it in certain families. Literature data suggest that the Asian BS (Saudi) haplotype may also be associated with unidentified high Hb- F determinants. Part of this proposal aims to clarify the relationship between haplotype and Hb F expression in SS and beta- thal. We will test the related hypotheses that certain occurrences of the Benin and Asian betaS haplotypes have closely-linked but unidentified high Hb F mutations. For selected cases (high Hb F Benin and Asian betaS), amplified genomic DNA will be sequenced in the G gamma and A gamma promoters. If no mutations are found, then the gamma enhancer will be tested in the Bodine & Ley expression system and sequenced if activity is elevated. We will also test the hypothesis that the 4 bp deletion is a major factor the severity of betao -thal by using oligonucleotide probes to screen mild and severe cases. The second part of this proposal will test the hypothesis that the -158, -161, -202 mutations and the 4 bp deletion act by altering affinity for transcription-regulating proteins. Our model suggests possible protein-binding sites at several sequence motifs, including "TC" (198 to -192), "G-C rich" (-208 to -200), SphI (- 226 to -219) and "enhancer core" (-270 to -264). Mutant promoters will be placed 5' to a "marked" gamma globin gene in the presence and absence of gamma enhancer, and expression tested in K562 erythroleukemia cells by mRNA assays. Deletions 5' to the -200 and -158 mutations will test the importance of 5' sequences to elevated expression of those mutations. Comparison of protein binding to these motifs in normal and mutant promoter fragments will be done by gel retardation assays. Competition between normal and mutant oligomers will show the effects of mutations on protein binding. Footprinting and methylation interference data will precisely define binding sites, test the hypothesis that the -158 and -161 mutations affect binding to known motifs, and aid in characterizing partially purified proteins of K562 and bone marrow erythroid cells. These data will contribute to the mapping of structure- function relationships of gamma globin transcription in red cells of human adults.

胎儿血红蛋白（Hb F）升高可改善成人疾病 血红蛋白，如镰状细胞性贫血（SS）和β-地中海贫血。 这个实验室已经证明了 在-158（C至T）时，体内γ基因表达增加， G γ基因的-161（G到A）和A γ基因的-202（C到T）。 在A γ基因的-222至-225处的4个碱基对缺失， 与地中海A γ表达减少相关 单倍型II β-地中海贫血。 贝宁β S单倍型通常与低Hb F相关， 但初步数据显示， 某些家庭。 文献数据表明，亚洲BS （沙特）单倍型也可能与不明的高Hb- F行列式。 本建议的一部分旨在澄清 单倍型与SS和β-Hb F表达的关系 塔尔。 我们将检验相关的假设， 贝宁和亚洲betaS单倍型有密切联系， 未鉴定的高Hb F突变。 对于选定的病例（高Hb F 贝宁和亚洲betaS），扩增的基因组DNA将在 G γ和A γ启动子。 如果没有发现突变， 伽马增强子将在Bodine & Ley表达中进行测试 系统和排序，如果活动是升高。 我们还将测试 假设4 bp缺失是一个主要因素， 应用寡核苷酸探针筛选β-塔尔严重程度 轻度和重度病例。 本提案的第二部分将检验以下假设： -158、-161、-202突变和4 bp缺失通过改变 对转录调节蛋白的亲和力。 我们的模型显示 在几个序列基序上的可能的蛋白质结合位点， 包括“TC”（198至-192）、“富含G-C”（-208至-200）、SphI（- 226至-219）和“增强子核心”（-270至-264）。 突变型启动子 将被放置在“标记的”γ珠蛋白基因的5'端， 和不存在γ增强子，并在K562中检测表达 红白血病细胞的mRNA测定。 -200和-200的5'缺失 -158突变将测试5'序列对升高的突变的重要性。 这些突变的表达。 蛋白质结合的比较 将在正常和突变启动子片段中的这些基序 通过凝胶阻滞测定。 正常和突变之间的竞争 寡聚体将显示突变对蛋白结合的影响。 足迹和甲基化干扰数据将精确地 定义结合位点，检验-158和-161 突变影响与已知基序的结合，并有助于表征 K562和骨髓红系细胞部分纯化蛋白 细胞 这些数据将有助于绘制结构图- 红细胞γ-珠蛋白转录的功能关系 成年人。